Optical imaging is a rapidly developing field of research aimed at non-invasively interrogating animals for disease
progression, determining the effects of a drug on a particular pathology, assessing the pharmacokinetic behavior of a
drug, or identifying molecular biomarkers of disease. One of the key components of molecular imaging is the
development of specific, targeted imaging contrast agents to assess these biological processes. The development of
robust fluorochrome-labeled optical agents is a process that is often underestimated in terms of its complexity. We
describe here the development process and performance issues for three different optical agents: IRDye 800CW EGF
(epidermal growth factor), IRDye(R) 800CW 2-DG (2-deoxy D-glucose), and an IRDye 680 BoneTagTM. In vitro
competitive assays were developed for two of the markers to demonstrate specificity. Specificity was confirmed in
animal studies. Uptake of IRDye 800CW 2-DG was also examined by near-infrared confocal microscopy. Histological
examinations were performed on target and non-target tissues following the completion of the imaging studies. The
issues unique to the development of each labeled marker are discussed.